Project Summary/Abstract Inflammatory Bowel Disease (IBD) is a global health burden currently affecting around 3 million people in the United States with increasing incidence worldwide. According to a recent report, prevalence of IBD among US veterans rapidly increased during the past decade. Despite all the advancement, pathogenesis of IBD is still unclear due to the multifactorial nature of the disease and the treatment options are not adequate. Therefore, understanding the molecular pathophysiology of IBD is critical for developing effective therapeutic modalities. Several different mechanisms likely account for the IBD pathogenesis. In this regard, compromised Intestinal barrier is one of the most critical early event linked to the onset of intestinal inflammation in IBD. Recent studies have implicated novel roles of epithelial ion transporters (e.g. NHE3 and CFTR) in maintaining barrier integrity. In this regard, DRA (Down Regulated in Adenoma or SLC26A3) is the key transporter mediating chloride absorption in the mammalian intestine. DRA KO mice exhibit lack of an adherent inner mucus layer, altered proliferative homeostasis of the colonic crypts, and are more susceptible to experimental colitis. The important role of DRA in inflammation is further evident from GWAS, identifying DRA as a novel IBD susceptibility gene. Further, DRA levels are severely depleted in human IBD patient colonic mucosa and in mouse models recapitulating IBD. However, the mechanisms underlying this increased susceptibility to inflammation in response to loss of DRA are not known and warrant detailed investigations. Our preliminary data provide strong evidence for a novel role of DRA in maintenance of epithelial integrity. Our preliminary data suggest that loss of DRA may play a key role in breaching the epithelial barrier via multiple mechanisms e.g. either by directly affecting the TJ/AJ and/or via indirect (related to its Cl-/HCO3- exchange function, its cytoskeletal interactions and/or microbial dysbiosis). In addition, targeting DRA via its upregulation appears to be a novel therapeutic approach to restore barrier function and alleviating inflammation. However, the therapeutic feasibility of targeting DRA to restore barrier function in preclinical models has never been investigated. Therefore, we hypothesize that i) DRA loss perturbs intestinal barrier function by reducing TJ and AJ protein expression via posttranscriptional mechanisms involving RNA binding proteins and/or via direct interactions with cytoskeletal elements. ii) Upregulation of DRA function and expression can alleviate inflammation via restoration of intestinal barrier function. Our hypothesis will be tested utilizing state-of-the-art approaches and human/mouse colonoids, Caco-2 cells and KO or transgenic mouse models. Two Specific Aims have been proposed: Aim 1. Elucidate the post-transcriptional mechanisms modulating gut barrier function and TJ/AJ proteins in response to DRA deficiency; and Aim 2. Elucidate the novel role of DRA upregulation in maintenance of intestinal epithelial integrity and tight junction proteins in experimental models of colitis and colonoids derived from healthy and IBD patients. Successful outcome of these studies should yield critical insights into the molecular mechanisms of intestinal TJ/AJ regulation and establish DRA as a novel therapeutic target to manage IBD. Additionally, these studies will provide the applicant a great training vehicle for becoming an expert in mechanisms underlying the novel role of a membrane chloride transporter in intestinal tight junction dysregulation and pathogenesis of IBD, as well as train him in cutting- edge approaches utilizing enteroids, colonoids and advanced imaging techniques to evolve him into an independent VA investigator focused on IBD research.